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    Rapid Commun. Mass Spectrom. 2004; 18: 25052512

    Published online in Wiley InterScience ( DOI: 10.1002/rcm.1636

    To the Editor-in-Chief


    Determination of Xipamide metabo-

    lite in human urine by high-perfor-

    mance liquid chromatography/diode-

    array detection, high-performance

    liquid chromatography/electrospray

    ionization mass spectrometry and gas

    chromatography/mass spectrometry

    Diuretic agents are drugs that increase

    renal excretion of water and solutes

    (mainly sodium salts). The major pur-

    poses of diuretic therapy are to

    decrease fluid volume of the body and

    to adjust the water and electrolyte bal-

    ance. Diuretics are drugs widely used

    in clinical practice mainly in the treat-

    ment of hypertension and in different

    kinds of edema.13

    Recently, diuretics have been abused

    in sports with weight classes, such as

    weightlifting, wrestling and boxing.

    Athletes try to reduce their body

    weight in order to qualify for lower

    weight classes. It is also reported that

    athletes use diuretics to avoid detection

    of doping agents by reducing their

    urine concentration.4 As part of their

    efforts to fight drug abuse in sports, the

    Medical Commission of the Interna-

    tional Olympic Committee has banned

    diuretics since the 1988 Seoul Olympic


    Diuretics may be classified accord-

    ing to their chemical structure, mecha-

    nism, primary site of action in the

    nephron and their diuretic potency.5

    These diuretics exert their effects by

    inhibiting tubular sodium and water

    reabsorption by epithelial cells lining

    the renal tubule system. Certain diure-

    tics (such as carbonic anhydrase inhi-

    bitors, loop diuretics, thiazide-like

    diuretics and potassium-sparing diure-

    tics) suppress sodium and water reab-

    sorption by inhibiting the function of

    specific proteins that are responsible

    Copyright # 2004 John Wiley & Sons, Ltd.


    Letter to the Editor

    Table 1. HPLC/DAD operating conditions

    HPLC conditionsFlow rate: 1.0 mL/minMobile phase solvent A: Phosphate buffer (pH 6.8)

    solvent B: AcetonitrileGradient timetable:Time (min) 0 10 15 18Solvent A (%) 96 70 55 50Solvent B (%) 4 30 45 50Detection wavelength: 232 nmInjection volume: 5mLColumn temperature: 408CColumn: Hypersil-ODS C18 column (4.6 mmi.d. 100 mm, particle size 5mm)

    Table 2. HPLC/ESI-MS operating conditions

    HPLC conditions

    Flow Rate: 0.2 mL/minMobile Phase solvent A: Acetonitrile

    solvent B: 20 mM Ammonium formate (pH 4)Gradient timetable:Time (min) 0 5 18Solvent A (%) 20 50 50Solvent B (%) 80 50 50Injection Volume: 5mLColumn Temperature: 408CColumn: Capcell Pak C18 column (MG type, 2.0 mmi.d. 150 mmparticle size 5mm)MS conditionsIonization: ESI (electrospray ionization)Mode: Positive ionMass range: m/z 50420Nebulizing Gas Pressure: 30 psi (N2)Drying Gas Temp: 3508CDrying Gas Flow: 8.0 L/minCapillary Exit Voltage: 60.0 eVMS/MS conditions

    Collision gas: He (70 psi)Collision energy: 0.8 eVIsolation width of parent ion: 1.5 Da

    Table 3. GC/MS operating conditions

    GC conditionsCarrier gas: He at 1.0 mL/minOven temp. program:

    Initial temp.(8C)

    Initialtime (min)


    Final temp.(8C)

    Final time(min)

    180 1 15 300 5

    Injection volume: 2mLSplit mode: Split (1:10)Injection port temp.: 2808CTransfer line temp.: 2808CColumn: Ultra-2 (cross-linked 5 %phenylmethylsiloxane, 0.2 mmi.d 17 m length 0.33mmfilm thickness)MS conditions

    Ionization: EISource temp.: 2008CAcquisition mode: scanMass range: m/z 50600

  • for (or participate in) the transportation

    of electrolytes across the epithelial

    membrane; osmotic diuretics inhibit

    water and sodium and water reabsorp-

    tion by increasing intratubular osmotic

    pressure. Different types of diuretics

    may inhibit different transporters in dif-

    ferent segments of the tubular system.6,7

    Xipamide (4-chloro-5-sulfamoyl-20,60-salicyloxylidide) is a diuretic drug

    used for the treatment of high blood

    pressure and a edema of cardiac,

    hepatic or renal origin. It is a non-

    thiazide diuretic with a greater anti-

    hypertensive effect and may cause a

    lower potassium loss relative to

    sodium excretion than these drugs.

    Xipamide offers a suitable alternative

    to other diuretics in the treatment of

    patients with mild to moderate hyper-

    tension and of patients with edema due

    to a variety of causes.8

    Metabolite monitoring is important

    for the determination of any medica-

    tion by drugs. Several chromatogra-

    phic methods have been reported for

    the separation, detection, and quanti-

    tative measurement of individual

    diuretic agents in biological fluids. Pub-

    lished methods include those based

    on thin-layer chromatography (TLC),9

    gas-liquid chromatography (GLC),10

    gas chromatography/mass spectro-

    metry (GC/MS),10,11 high-performance

    liquid chromatography (HPLC),1216

    and high-performance liquid chroma-

    tography/mass spectrometry (HPLC/

    MS).17 Generally, metabolites in urine

    are polar, so it is not easy to use GC/MS

    without derivatization. More recently,

    HPLC with detection by tandem mass

    spectrometry (HPLC/MS/MS) has

    been used for trace level bioanalysis.18

    This technique allows highly sensitive

    determinations without the need for

    derivatization as required for GC/MS.

    It is also possible to devise methods

    specific for metabolites and structurally

    similar compounds.

    This paper describes the HPLC/

    diode-array detection (DAD) screening

    procedure and HPLC/MS confirma-

    tion method of Xipamide in human

    urine for doping control tests. The

    metabolite of Xipamide was also stu-

    died by GC/MS after methylation for


    Xipamide tablets were purchased

    from Bukwang Pharm. Co., Ltd. (Seoul,

    Korea). Anhydrous sodium sulfate was

    Scheme 1. Structure of Xipamide.

    Figure 1. HPLC/DAD chromatograms of Xipamide in (a) spiked urine, (b) blank

    urine, and (c) dosed urine.

    Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512

    2506 Letter to the Editor

  • purchased from Sigma Chemical Co.

    (MO, USA) and HCl was from Merck

    (Darmstadt, Germany). Diethyl ether,

    ethyl acetate and acetone were HPLC

    grade from J. T. Baker (Phillipsburg,

    NJ, USA). Acetonitrile and methanol

    from J. T. Baker and distilled water

    were used after filtering through Milli-

    pore filters (0.5 and 0.45 mm, respec-tively) and sonication for 20 min. The

    internal standard (ethyltheophylline)

    was prepared at the Doping Control

    Center (KIST, Korea)

    A Hewlett-Packard (Palo Alto, CA,

    USA) HP 1100 series liquid chromato-

    graph, coupled with a HP 1100 series

    G1315A diode-array detector, was

    used to screen Xipamide and its meta-

    bolite. The column for HPLC was a

    Hypersil-ODS C18 column (4.6 mm

    i.d. 100 mm, particle size 5mm, Ther-mohypersil). The HPLC/ESI-MS sys-

    tem consisted of an HP 1100 series

    binary pump HPLC system (Agilent,

    Palo Alto, CA, USA) and an LC/MSD

    ion trap equipped with an electrospray

    ionization (ESI) source. It was used for

    confirmation of Xipamide and charac-

    terization of suspected metabolites.

    The column for HPLC/ESI-MS was a

    Capcell Pak C18 column (MG type,

    2.0 mm i.d. 150 mmparticle size5 mm, Shiseido). A Trace GC-Polaris Q(Finnigan Inc., San Jose, CA, USA) was

    used for reconfirmation of the suspec-

    ted metabolite. The column for GC/

    MS was an Ultra-2 capillary column

    (0.2 mm i.d. 17 m length 0.33 mmfilm thickness, Agilent Technologies).

    A Lauda (Lauda-Konigshofen, Ger-

    many) Ecoline RE112 freezer was used

    to freeze the aqueous layer. A Turbo-

    vap1 LV evaporator supplied by

    Zymark Corporation (Hopkinton,

    MA, USA) was used to evaporate the

    extracted organic solvents.

    A healthy male volunteer (age 29,

    weight 70 kg) was dosed with one

    Diurexan tablet (Xipamide, 20 mg/

    tab). Urine samples were collected for

    48 h and subjected to the screening

    procedure; these urine samples were

    kept at 238C.Anhydrous sodium sulfate (1 g) was

    added to the urine (pH 5 adjusted with

    phosphate buffer). After vortexing,

    5 mL distilled diethyl ether were

    added. After shaking for 20 min and

    centrifugation at 2500 rpm for 5 min,

    the solution was frozen in the freezer

    (308C). The organic phase was trans-ferred to another tube and evaporated

    to dryness at 408C under a gentlestream of nitrogen. The residue was

    then reconstituted with 200 mL metha-nol, of which 5 mL were injected on theHPLC/DAD and LC/MS system.

    Then, 150mL of the residue were driedunder a nitrogen gas stream, and 50mLof methyl iodide and 150 mL of acetonewere added and then vortex-mixed.

    The mixture was heated at 808C for 1 hand then cooled to room temperature.

    Finally, 2 mL of this solution wereanalyzed by the GC/MS system.

    Operating conditions for HPLC/

    DAD and HPLC/ESI-MS, used for the

    detection of Xipamide and the char-

    acterization of its metabolite, are

    described in Tables 1 and 2. Mass

    spectrometry (MS) and tandem mass

    spectrometry (MS/MS) analyses were



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